Treatment for central nervous system disorders

ABSTRACT

Compositions that include an Aβ polypeptide linked to a non-Aβ polypeptide are described, as well as methods of using such compositions.

TECHNICAL FIELD

[0001] This invention relates to compositions for treating centralnervous system (CNS) disorders such as Alzheimer's disease (AD), andmore particularly, to compositions that contain a β amyloid (Aβ)polypeptide linked to a non-Aβ polypeptide.

BACKGROUND

[0002] Both active and passive immunization involving Aβ-peptides orspecific monoclonal antibodies against these peptides have been assessedfor the treatment and prevention of AD. Reducing Aβ accumulation byactive immunization improves cognitive performance in mice. See, forexample, Chen et al., Nature, 408:975-979 (2000); Janus et al. Nature,408:979-982 (2000); and Morgan et al., Nature, 408:982-985 (2000). Themechanism by which host-generated antibodies against Aβ clear brainsenile plaques is far from being understood. Active immunizationexperiments use complete Freund's adjuvant, which, by itself, inducesleakage of serum proteins, including IgG, through the blood-brainbarrier (BBB) 2-3 weeks after injection and cannot be used as anadjuvant in humans. Passive immunization studies are confounded by theintegrity of the BBB, which restricts passage of immunoglobulins. Thepermeability coefficient×surface area (PS) product of IgG has beenquantified in rats and found to be very low (0.03-0.1×10⁻⁶ mg/g/sec) andis consistent with a transport mechanism of passive diffusion orfluid-phase endocytosis.

SUMMARY

[0003] The invention is based on the discovery that Aβ-immune complexesare transported across the BBB via a receptor-mediated process at a rategreater than that of antibody alone. Thus, transport of antibodieshaving specific binding affinity for Aβ across the BBB, or otherpolypeptides that have low permeability at the BBB, can be enhanced whenlinked to an Aβ polypeptide. As a result, the success of passiveimmunization and therapy for AD as well as other CNS disorders isenhanced. Polyamine modified antibodies having specific binding affinityfor Aβ also have increased permeability at the BBB and can be used forpassive immunization and treatment of AD.

[0004] In one aspect, the invention features a composition that includesan Aβ polypeptide and a non-Aβ polypeptide, wherein the Aβ polypeptideand the non-Aβ polypeptide are linked (e.g., covalently). Thecomposition further can include a pharmaceutically acceptable carrier orexcipient. The non-Aβ polypeptide can be an antibody or a fragmentthereof (e.g., a Fab fragment, a single chain Fv antibody fragment, or aF(ab)₂ fragment). The antibody can be labeled with a radioisotope or acontrast agent. The antibody can have specific binding affinity foramyloid. The non-Aβ polypeptide also can be an enzyme such as anantioxidant enzyme (e.g., catalase or superoxide dismutase), a cytokinesuch as an interferon, an interleukin, or a neurotrophic factor, orleptin. The Aβ polypeptide can include residues 1-40, 1-42, or 1-43 ofSEQ ID NO:1.

[0005] The invention also features a method of treating a patientdiagnosed with AD. The method includes administering to the patient anamount of a composition effective to treat AD, wherein the compositionincludes an Aβ polypeptide and an antibody having specific bindingaffinity for the Aβ polypeptide. The antibody can be a Fab fragment, asingle chain Fv antibody fragment, or a F(ab)₂ fragment.

[0006] In another aspect, the invention features a method of treating apatient diagnosed with AD. The method includes administering to thepatient an amount of an antibody effective to treat AD, wherein theantibody is polyamine modified and has specific binding affinity for anAD polypeptide.

[0007] In yet another aspect, the invention features a method ofdiagnosing AD in a patient. The method includes administering acomposition to the patient, wherein the composition includes an Aβpolypeptide and an antibody having specific binding affinity foramyloid, wherein the antibody is labeled, and detecting the presence orabsence of the antibody bound to amyloid in the brain of the patient,wherein the patient is diagnosed with AD based on the presence oflabeled amyloid (e.g., labeled amyloid deposits such as β-amyloidplaques). The detecting step can include diagnostic imaging (e.g.,positron emission tomography, gamma-scintigraphy, single photon emissioncomputerized tomography, magnetic resonance imaging, functional magneticresonance imaging, or magnetoencephalography). Magnetic resonanceimaging is particularly useful. The antibody can be labeled with acontrast agent (e.g., gadolinium, dysprosium, or iron). Gadolinium is aparticularly useful contrast agent.

[0008] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used topractice the invention, suitable methods and materials are describedbelow. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

[0009] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DETAILED DESCRIPTION

[0010] The invention features compositions containing Aβ polypeptidesthat can be used to enhance transport of non-Aβ polypeptides across theBBB. As described herein, BBB permeability of a composition containingAβ bound to a monoclonal antibody was significantly greater than that ofthe monoclonal antibody alone. Without being bound by a particularmechanism, Aβ itself may be responsible for transporting the antibodyacross the BBB. Thus, Aβ can be used to enhance the permeability ofother polypeptides at the BBB, and as a result, compositions of theinvention can be used in the diagnosis, treatment, and/or prevention ofneurodegenerative disorders such as AD, Parkinson's disease,frontotemporal dementias (e.g., Pick's disease), and amyloidoticpolyneuropathies, transmissible spongiform encephalopathies (i.e., priondiseases) such as Creutzfeldt-Jakob disease (CJD),Gerstmann-Straiussler-Scheinker syndrome, and fatal familial insomnia,demyelinating diseases such as multiple sclerosis, and amyotropiclateral sclerosis.

[0011] Aβ Compositions

[0012] Compositions of the invention include a purified Aβ polypeptidelinked to a purified non-Aβ polypeptide. As used herein, the term“purified” refers to a polypeptide that is separated from cellularcomponents (e.g., other polypeptides, lipids, carbohydrates, and nucleicacids) that are naturally associated with the polypeptide. Thus, apurified polypeptide is any polypeptide that is removed from its naturalenvironment and is at least 75% pure (e.g., at least about 80, 85, 90,95, or 99% pure). Typically, a purified polypeptide will yield a singlemajor band on a non-reducing polyacrylamide gel.

[0013] As used herein, “Aβ polypeptide” refers to 1) the naturallyoccurring human Aβ polypeptide (DAEFRHDSGY EVHHQKLVFF AEDVGSNKGAIIGLMVGGVV IAT, SEQ ID NO:1) 2) polypeptides having one or moresubstitutions or insertions in the amino acid sequence of the naturallyoccurring human Aβ polypeptide that retain the ability to cross the BBB,and 3) fragments of 1) and 2) that retain the ability to cross the BBB.Permeability of an Aβ polypeptide at the BBB can be assessed accordingto the methods of Example 1. See also Poduslo et al., Proc. Natl. Acad.Sci USA 89:2218-2222 (1992) and Poduslo et al., Neurobiol. Disease8:555-567 (2001). The naturally-occurring human Aβ polypeptide ranges inlength from 39 to 43 amino acids (residues 1 to 39, 1 to 40, 1 to 41, 1to 42, or 1 to 43 of SEQ ID NO:1) and is a proteolytic cleavage productof the amyloid precursor protein (APP). Non-limiting examples of aminoacid substitutions that can be introduced into human Aβ includesubstitutions at amino acid residues 5, 10, 13, 19, and 20 of SEQ IDNO:1, or combinations thereof. In particular, a glycine can besubstituted for the arginine at residue 5, a phenylalanine can besubstituted for the tyrosine at residue 10, or an arginine can besubstituted for the histidine at residue 13. Such substitutions do notalter the properties of human Aβ polypeptide. See Fraser et al.,Biochemistry 31:10716-10723 (1992); and Hilbich et al., Eur. J. Biochem.201:61-69 (1992). An isoleucine, leucine, threonine, serine, alanine,valine, or glycine can be substituted for the phenylalanine residues atpositions 19 and 20.

[0014] Suitable fragments of Aβ polypeptides are about 6 to 38 aminoacid residues in length (e.g., 10 to 36, 10 to 34, 10 to 30, 12 to 28,14 to 26, 16 to 24, or 18 to 22 amino acid residues in length) andretain the ability to cross the BBB. For example, an Aβ polypeptide maycontain residues 1 to 10, 1 to 15, 1 to 20, 5 to 15, 5 to 20, 5 to 25,10 to 20, 10 to 25, 10 to 30, 15 to 25, 15 to 30, or 15 to 35 of SEQ IDNO:1. Alternatively, an Aβ polypeptide may include residues 20 to 30, 20to 35, 20 to 40, 25 to 35, 25 to 40, 30 to 40, 25 to 42, or 30 to 42 ofSEQ ID NO:1.

[0015] Aβ polypeptides can be linked to non-Aβ polypeptides via covalentlinks. Covalent cross-linking techniques are known in the art. See, forexample, “Chemistry of Protein Conjugation and Cross-Linking”, Shan S.Wong, CRC Press, Ann Arbor, 1991. Suitable cross-linking reagents do notinterfere with the binding of the Aβ polypeptide to its cognate receptorand are chosen for appropriate reactivity, specificity, spacer armlength, membrane permeability, cleavability, and solubilitycharacteristics. Similarly, suitable cross-linking reagents do notinterfere with binding of a non-Aβ polypeptide to its binding partner(e.g., cognate receptor or epitope on a macromolecule). Cross-linkingreagents are available commercially from many sources including PierceChemical Co., Rockford, Ill.

[0016] An Aβ polypeptide and a non-Aβ polypeptide can be covalentlycross-linked using, for example, glutaraldehyde, a homobifunctionalcross-linker, or a heterobifunctional cross-linker. Glutaraldehydecross-links polypeptides via their amino moieties. Homobifunctionalcross-linkers (e.g., a homobifunctional imidoester, a homobifunctionalN-hydroxysuccinimidyl (NHS) ester, or a homobifunctional sulfhydrylreactive cross-linker) contain two or more identical reactive moietiesand can be used in a one step reaction procedure in which thecross-linker is added to a solution containing a mixture of thepolypeptides to be linked. Homobifunctional NHS esters and imido esterscross-link amine containing polypeptides. In a mild alkaline pH, imidoesters react only with primary amines to form imidoamides, and overallcharge of the cross-linked polypeptides is not affected.Homobifunctional sulfhydryl reactive cross-linkers includebismaleimidhexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and1,4-di-(3′,2′-pyridyldithio) propionamido butane (DPDPB).

[0017] Heterobifunctional cross-linkers have two or more differentreactive moieties (e.g., an amine reactive moiety and asulfhydryl-reactive moiety) and are cross-linked with one of thepolypeptides via the amine or sulfhydryl reactive moiety, then reactedwith the other polypeptide via the non-reacted moiety. Multipleheterobifunctional. haloacetyl cross-linkers are available, as arepyridyl disulfide cross-linkers. Carbodiimides are a classic example ofheterobifunctional cross-linking reagents for coupling carboxyls toamines, which results in an amide bond.

[0018] Alternatively, an Aβ polypeptide can be linked to a non-Aβpolypeptide such as an antibody via the specific binding affinity of theantibody for the Aβ polypeptide. Purified Aβ polypeptide and antibodycan be incubated together at 37° C. in an appropriate buffer (e.g.,phosphate buffered saline) to form an immune complex. Such an immunecomplex constitutes a composition of the invention.

[0019] Aβ polypeptides can be linked to any non-Aβ polypeptide, and inparticular, to any polypeptide that is useful for diagnosing or treatinga disorder of the CNS. Non-Aβ polypeptides are at least six amino acidresidues in length. For example, an Aβ polypeptide can be linked to anenzyme such as an antioxidant enzyme, which can protect cells againstreactive oxygen species. Non-limiting examples of antioxidant enzymesinclude catalase (E.C. 1.11.1.6), superoxide dismutase (E.C. 1.15.1.1),glutathione peroxidase (E.C. 1.6.4.2), and glutathione reductase (E.C.1.11.1.9).

[0020] Aβ polypeptides also can be linked to cytokines such as aninterferon (e.g., interferon α, β, or γ), interleukin (IL) (e.g., IL-1aor b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, orIL-12), neurotrophic factors such as neurotrophins (e.g., nerve growthfactor or brain-derived neurotrophic factor), neuropoietic factors suchas cholinergic differentiation factor, ciliary neurotrophic factor,oncostatin M, growth-promoting factor, and sweat gland factor, andgrowth factor peptides such as glial-cell line-derived neurotrophicfactor, or a hormone such as leptin.

[0021] In addition, Aβ polypeptides can be linked to an antibody. Forexample, an Aβ polypeptide can be linked to an antibody having specificbinding affinity for amyloid deposits of Aβ or of a prion protein (PrP).See U.S. Pat. No. 5,231,000 and U.S. Pat. No. 5,262,332 for examples ofantibodies having specific binding affinity for Aβ. See Zanusso et al.,Proc. Natl. Acad. Sci. USA, 95:8812-8816 (1998) for examples ofantibodies having specific binding affinity for the protease resistantform of PrP. As used herein, the term “antibodies” includes polyclonalor monoclonal antibodies, humanized or chimeric antibodies, and antibodyfragments such as single chain Fv antibody fragments, Fab fragments, andF(ab)₂ fragments. Monoclonal antibodies are particularly useful. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region. Chimeric antibodies can be produced through standardtechniques.

[0022] Antibody fragments can be generated by known techniques. Forexample, F(ab′)₂ fragments can be produced by pepsin digestion of theantibody molecule, and Fab fragments can be generated by reducing thedisulfide bridges of F(ab′)₂ fragments. Alternatively, Fab expressionlibraries can be constructed. See, for example, Huse et al., Science,246:1275 (1989). Single chain Fv antibody fragments are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge (e.g., 15 to 18 amino acids), resulting in a singlechain polypeptide. See, for example, U.S. Pat. No. 4,946,778.

[0023] In some embodiments, the Aβ polypeptide and/or the non-Aβpolypeptide are labeled to facilitate diagnosis of a CNS disorder.Typical labels that are useful include radioisotopes and contrast agentsused for imaging procedures in humans. Non-limiting examples of labelsinclude radioisotope such as ¹²³¹I (iodine), ¹⁸F (fluorine), ^(99m)Tc(technetium), ¹¹¹In (indium), and ⁶⁷Ga (gallium), and contrast agentssuch as gadolinium (Gd), dysprosium, and iron. Radioactive Gd isotopes(¹⁵³Gd) also are available and suitable for imaging procedures innon-human mammals. Polypeptides can be labeled through standardtechniques. For example, polypeptides can be iodinated using chloramineT or 1,3,4,6-tetrachloro-3α,6α-diphenylglycouril. For fluorination,polypeptides are synthesized and fluorine is added during the synthesisby a fluoride ion displacement reaction. See, Muller-Gartner, H., TIBTech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug CarrierSyst., 16(2):209-244 (1999) for a review of synthesis of proteins withsuch radioisotopes.

[0024] Polypeptides also can be labeled with a contrast agent throughstandard techniques. For example, polypeptides can be labeled with. Gdby conjugating low molecular Gd chelates such as Gd diethylene triaminepentaacetic acid (GdDTPA) or Gd tetraazacyclododecanetetraacetic(GdDOTA) to the polypeptide. See, Caravan et al., Chem. Rev.99:2293-2352 (1999) and Lauffer et al. J. Magn. Reson. Imaging 3:11-16(1985). Antibodies can be labeled with Gd by, for example, conjugatingpolylysine-Gd chelates to the antibody. See, for example, Curtet et al.,Invest. Radiol. 33(10):752-761 (1998). Alternatively, antibodies can belabeled with Gd by incubating paramagnetic polymerized liposomes thatinclude Gd chelator lipid with avidin and biotinylated antibody. See,for example, Sipkins et al. Nature Med., 4 623-626 (1998).

[0025] Nucleic Acids Encoding Aβ and Non-Aβ Polypeptides

[0026] Isolated nucleic acid molecules encoding Aβ and non-Aβpolypeptides of the invention can be produced by standard techniques. Asused herein, “isolated” refers to a sequence corresponding to part orall of a gene encoding an Aβ or non-Aβ polypeptide, but free ofsequences that normally flank one or both sides of the wild-type gene ina mammalian genome. An isolated nucleic acid can be, for example, arecombinant DNA molecule, provided one or both of the nucleic acidsequences normally found immediately flanking that DNA molecule in anaturally-occurring genome is removed or absent. Thus, isolated nucleicacids include, without limitation, a DNA that exists as a separatemolecule (e.g., a cDNA or genomic DNA fragment produced by PCR orrestriction endonuclease treatment) independent of other sequences aswell as recombinant DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,adenovirus, or herpes virus), or into the genomic DNA of a prokaryote oreukaryote. In addition, an isolated nucleic acid can include arecombinant DNA molecule that is part of a hybrid or fusion nucleicacid. A nucleic acid existing among hundreds to millions of othernucleic acids within, for example, cDNA or genomic libraries, or gelslices containing a genomic DNA restriction digest, is not to beconsidered an isolated nucleic acid.

[0027] Isolated nucleic acid molecules are at least about 18 nucleotidesin length. For example, the nucleic acid molecule can be about 18 to 20,20-50, 50-100, or greater than 150 nucleotides in length. Nucleic acidmolecules can be DNA or RNA, linear or circular, and in sense orantisense orientation.

[0028] Specific point changes can be introduced into the nucleic acidsequence encoding the naturally-occurring human Aβ polypeptide by, forexample, oligonucleotide-directed mutagenesis. In this method, a desiredchange is incorporated into an oligonucleotide, which then is hybridizedto the wild-type nucleic acid. The oligonucleotide is extended with aDNA polymerase, creating a heteroduplex that contains a mismatch at theintroduced point change, and a single-stranded nick at the 5′ end, whichis sealed by a DNA ligase. The mismatch is repaired upon transformationof E. coli or other appropriate organism, and the gene encoding themodified vitamin K-dependent polypeptide can be re-isolated from E. colior other appropriate organism. Kits for introducing site-directedmutations can be purchased commercially. For example, Muta-Gene®in-vitro mutagenesis kits can be purchased from Bio-Rad Laboratories,Inc. (Hercules, Calif.).

[0029] Polymerase chain reaction (PCR) techniques also can be used tointroduce mutations. See, for example, Vallette et al., Nucleic AcidsRes., 17(2):723-733 (1989). PCR refers to a procedure or technique inwhich target nucleic acids are amplified. Sequence information from theends of the region of interest or beyond typically is employed to designoligonucleotide primers that are identical in sequence to oppositestrands of the template to be amplified, whereas for introduction ofmutations, oligonucleotides that incorporate the desired change are usedto amplify the nucleic acid sequence of interest. PCR can be used toamplify specific sequences from DNA as well as RNA, including sequencesfrom total genomic DNA or total cellular RNA. Primers are typically 14to 40 nucleotides in length, but can range from 10 nucleotides tohundreds of nucleotides in length. General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Ed. by Dieffenbach, C.and Dveksler, G., Cold Spring Harbor Laboratory Press, 1995.

[0030] Nucleic acids encoding Aβ and non-Aβ polypeptides also can beproduced by chemical synthesis, either as a single nucleic acid moleculeor as a series of oligonucleotides. For example, one or more pairs oflong oligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a double-stranded nucleicacid molecule per oligonucleotide pair, which then can be ligated into avector.

[0031] Producing Purified Polypeptides

[0032] Purified Aβ and non-Aβ polypeptides of the invention can beobtained from commercial sources, or alternatively, can be obtained byextraction from a natural source (e.g., liver tissue), chemicalsynthesis, or by recombinant production in a host cell. In general,recombinant polypeptides are produced by introducing an expressionvector that contains a nucleic acid encoding the polypeptide of interestoperably linked to regulatory elements necessary for expression of thepolypeptide into a bacterial or eukaryotic host cell (e.g., insect,yeast, or mammalian cells). Regulatory elements do not typically encodea gene product, but instead affect the expression of the nucleic acidsequence. In bacterial systems, a strain of Escherichia coli such asBL-21 can be used. Suitable E. coli vectors include the pGEX series ofvectors that produce fusion proteins with glutathione S-transferase(GST). Transformed E. coli are typically grown exponentially thenstimulated with isopropylthiogalactopyranoside (IPTG) prior toharvesting. Such fusion proteins typically are soluble and can bepurified easily from lysed cells by adsorption to glutathione-agarosebeads followed by-elution in the presence of free glutathione. The pGEXvectors are designed to include thrombin or factor Xa protease cleavagesites so that the cloned target gene product can be released from theGST moiety.

[0033] In eukaryotic host cells, a number of viral-based expressionsystems can be utilized to produce the polypeptides of interest. Anucleic acid encoding a polypeptide of the invention can be cloned into,for example, a baculoviral vector such as pBlueBac (Invitrogen, SanDiego, Calif.) and then used to co-transfect insect cells such asSpodoptera frugiperda (Sf9) cells with wild type DNA from Autographacalifornica multinuclear polyhedrosis virus (AcMNPV). Recombinantviruses producing polypeptides of the invention can be identified bystandard methodology. Alternatively, a nucleic acid encoding apolypeptide of the invention can be introduced into a SV40, retroviral,or vaccinia based viral vector and used to infect suitable host cells.

[0034] Mammalian cell lines that stably express a polypeptide ofinterest can be produced using an expression vector that contains aselectable marker and standard techniques. For example, the eukaryoticexpression vector pCR3.1 (Invitrogen, San Diego, Calif.) can be used toexpress polypeptides of interest in, for example, Chinese hamster ovary(CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3cells, BHK21 cells, MDCK cells, and human vascular endothelial cells(HUVEC). Following introduction of the expression vector byelectroporation, lipofection, calcium phosphate or calcium chlorideco-precipitation, DEAE dextran, or other suitable transfection method,stable cell lines are selected, e.g., by antibiotic resistance to G418,kanamycin, or hygromycin. Alternatively, a nucleic acid encoding thepolypeptide of interest can be ligated into a mammalian expressionvector such as pcDNA3 (Invitrogen, San Diego, Calif.) then transcribedand translated in vitro using wheat germ extract or rabbit reticulocytelysate.

[0035] Polypeptides of interest can be purified by known chromatographicmethods including DEAE ion exchange, gel filtration, and hydroxylapatitechromatography Polypeptides can be “engineered” to contain an amino acidsequence that allows the polypeptide to be captured onto an affinitymatrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine,or Flag™ tag (Kodak) can be used to aid polypeptide purification. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino termini. Other fusions that could be usefulinclude enzymes that aid in the detection of the polypeptide, such asalkaline phosphatase. Immunoaffinity chromatography also can be used topurify polypeptides of interest.

[0036] Polyamine Modified Antibodies

[0037] As described herein, polyamine modification of an antibody havingspecific binding affinity for Aβ enhances permeability of the modifiedantibody at the BBB. In particular, polyamine-modified monoclonalantibody against Aβ has a PS product that is 36 fold higher in thecortex compared to unmodified antibody and may provide a better approachto passive immunization for AD. Antibodies having specific bindingaffinity for Aβ can be modified with polyamines that are eithernaturally occurring or synthetic. See, for example, U.S. Pat. No.5,670,477. Useful naturally occurring polyamines include putrescine,spermidine, spermine, 1,3-diaminopropane, norspermidine,syn-homospermidine, thermine, thermospermine, caldopentamine,homocaldopentamine, and canavalmine. Putrescine, spermidine, andspermine are particularly useful. Synthetic polyamines are composed ofthe empirical formula C_(x)H_(y)N_(z), and can be cyclic or acyclic,branched or unbranched, hydrocarbyl chains of 3-12 carbon atoms thatfurther include 1-6 NR or N(R)₂ moieties, wherein R is H, (C₁-C₄) alkyl,phenyl, or benzyl. Polyamines can be linked to an antibody using thecross-linking techniques described above.

[0038] Diagnosis or Treatment of a CNS Disorder

[0039] Compositions of the invention can be formulated with apharmaceutically acceptable carrier and administered to a mammal. Forexample, a composition of the invention can be administered to anon-human animal (e.g., a transgenic mouse model of Alzheimer'sdisease). or to a human to aid in the diagnosis of a CNS disorder suchas Alzheimer's disease or for treating a human patient that has beendiagnosed with a CNS disorder. As used herein, the term “treatment” or“treating” refers to administering a composition of the invention to apatient, regardless of whether the patient responds to the treatment,with the proviso that when the same composition is administered to apopulation of patients, a statistically significant number of patientswithin the population exhibit a clinically recognized improvement orstabilization of one or more clinical features of the disorder.

[0040] In general, compositions of the invention are administeredintravenously (i.v.), although other parenteral routes ofadministration, including subcutaneous, intramuscular, intra-arterial,intranasal, intracarotid, and intrathecal also can be used. Formulationsfor parenteral administration may contain pharmaceutically acceptablecarriers such as sterile water or saline, polyalkylene glycols such aspolyethylene glycol, vegetable oils, hydrogenated naphthalenes, and thelike.

[0041] The dosage of the composition to be administered can bedetermined by the attending physician taking into account variousfactors known to modify the action of drugs. These include healthstatus, body weight, sex, diet, time and route of administration, othermedications, and any other relevant clinical factors. Typically, thedosage is about 1-3000 μg/kg body weight (e.g., from about 10-1000 μg/kgbody weight or 50-500 μg/kg body weight). Therapeutically effectivedosages may be determined by either in vitro or in vivo methods.

[0042] Treatment of a CNS disorder can be assessed by determining if oneor more clinical features of the disorder (e.g., cognitive function,memory, behavior, language skills, motor skills, or rigidity of thepatient) improve or are stabilized in the patient.

[0043] For diagnosis of a CNS disorder, the composition that isadministered to the patient contains at least one polypeptide that islabeled as described above. Presence or absence of the labeledpolypeptide (e.g., labeled antibody or labeled Aβ polypeptide) isdetected in the CNS in vivo (e.g., in the brain of the patient) using,for example, imaging techniques such as positron emission tomography(PET), gamma-scintigraphy, magnetic resonance imaging (MRI), functionalmagnetic resonance imaging (FMRI), magnetoencephalography (MEG), andsingle photon emission computerized tomography (SPECT). MRI isparticularly useful as the spatial resolution and signal-to-noise ratioprovided by MRI (30 microns) is suitable for detecting amyloid deposits,which can reach up to 200 microns in size. The CNS disorder can bediagnosed based on the presence, for example, of labeled amyloid (e.g.,labeled amyloid deposits).

[0044] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Materials and Methods

[0045] Aβ Proteins: Human Aβ₁₋₄₂ was synthesized by using f-mocchemistry in a Perkin-Elmer peptide synthesizer in the Mayo Protein CoreFacility. The amino acid sequence of human Aβ is provided in SEQ IDNO:1. Purity of the peptide was evaluated by peptide sequencing andlaser desorption mass spectrometry.

[0046] Monoclonal Antibody Generation: B-cell hybridomas were generatedfollowing the procedure of St. Groths and Scheidegger (J. Immunol.Methods 35:1 (1980)) in the Mayo Monoclonal Core Facility. Human Aβ₁₋₄₂that was aggregated and fibrilized by incubating at 37° C. for 24 hourswas used as antigen. Positive subclones were isotyped and cryopreservedand further characterized by ELISA and immunohistochemistry labeling ofAD transgenic mouse brain sections. A non-specific, monoclonal antibodywas obtained from ATCC HB96 L227 (anti-human Ia).

[0047] PS/V_(p) Measurements at the BBB for Radioiodinated MonoclonallgG (MoIgG): Aliquots of the proteins (MoIgG or Aβ) were labeled with¹²⁵I or ¹³¹I using the chloramnine T procedure described by Poduslo etal., Proc. Natl. Acad. Sci. USA 9:5705-5709 (1994). PS/V_(p)measurements were performed as described by Poduslo et al., Neurobiol.Diseases, 8:555-567 (2001) and Poduslo et al., Proc. Natl. Acad. Sci.USA 89:2218-2222 (1992). The procedure for quantifying BBB permeabilityof proteins was adapted from the rat to the mouse and includedcatheterizing the femoral artery and vein of the mouse instead of thebrachial artery and vein as for the rat. Because of the smaller bloodvolume in the mouse, serial sampling of 20 μl of blood from the femoralartery was performed and directly TCA precipitated to generate a wholeblood washout curve for the intact protein. Briefly, an I.V. bolusinjection of phosphate-buffered saline (PBS) containing ¹²⁵I-MoIgG (100μC) was rapidly injected into the femoral vein inpentobarbital-anesthetized mice. Serial blood samples were collectedfrom the femoral artery over the next 30-120 minutes. At 30-60 secondsbefore the end of the experiment, the second isotope of radiolabeledprotein (¹³¹I-MoIgG) (100 μC) was administered intravenously to serve asa V_(p) indicator.

[0048] After the final blood sample, the animals were sacrificed, thebrain and meninges were removed, and the brain was dissected into thecortex, caudate-putamen (neostriatum), hippocampus, thalamus, brainstem, and cerebellum. Tissue was lyophilized, and dry weights weredetermined with a microbalance and converted to respective wet weightswith wet weight/dry weight ratios previously determined. Tissue andplasma samples were assayed for ¹²⁵I and ¹³¹I radioactivity in atwo-channel gamma counter (Packard COBRA II) with radioactivitycorrected for crossover of ¹³¹I activity into the ¹²⁵I channel andbackground. Data are presented as {overscore (x)}±SEM values withstatistical evaluation using ANOVA with significance accepted at theP<0.05 level. The V_(p) and PS measurements were calculated as describedby Poduslo et al., Neurobiol. Disease, 8:555-567 (2001) and Poduslo etal., Proc. Natl. Acad. Sci. USA 89:2218-2222 (1992). All procedures wereperformed using humane and ethical protocols approved by the Mayo ClinicInstitutional Animal Care and Use Committee, in accordance with theNational Institute of Health's Guide for the Care and Use of LaboratoryAnimals. All efforts were made to minimize both the suffering and thenumber of animals used.

[0049] Immune Comlex Preparation: Human Aβ42 was incubated with itsradioiodinated monoclonal antibody (PC2) or the radioiodinatednon-specific monoclonal antibody (L227) for 1 hour at 37° C. in PBS atmole ratios of 10:1, 100:1, or 1000:1. Aliquots were then injected intothe femoral vein as an I.V. bolus.

[0050] Polyamine Modification of Monoclonal IgG: Modification of themonoclonal antibody (PC2) was performed as described by Poduslo andCurran, Proc.Natl. Acad. Sci. USA 89:2218-2222 (1992) and Poduslo andCurran, J. Neurochem. 66:1599-1609 (1996). Putrescine (PUT) wascovalently attached to carboxylic acids using carbodiimide. Ionizationof the carboxylic acid groups was controlled by pH, which in turncontrolled the extent of modification with the polyamine.

Example 2 Enhanced Permeability of Polyamine Modified Antibody andImmune Complexes at the BBB

[0051] The BBB permeability of a non-specific monoclonal antibody(anti-human Ia; L227; IgG₁κ), monoclonal antibody against human Aβ₁₋₄₂(PC2; IgG₁κ), and the immune complex [(human Aβ42)-L227 or (humanAβ42)-PC2)] at various mole ratios was determined in the normal adultmouse (B6SJL) as described in Example 1 by quantifying the permeabilitycoefficient×surface area (PS) product for each protein after correctionfor the residual plasma volume (V_(p)) occupied by the protein in bloodvessels in different brain regions following an I.V. bolus injection. Inthese experiments, the V_(p) value was determined with a second aliquotof the same protein radioiodinated with a different isotope of iodine(¹²⁵I vs. ¹³¹I) given 30-60 seconds before the end of the experiment.Using the same test substance allows for an accurate determination ofthe V_(p) and corrects for non-specific adherence to capillary walls,which would be characteristic of the protein tested. Similarly, a dualisotope approach allows for the determination of the vascular space ineach individual animal. The PS product at the BBB for differentradioiodinated proteins is corrected, therefore, for the V_(p) with asecond tracer of the same protein.

[0052] The PS product for the non-specific monoclonal antibody (L227)ranged from 0.5-1.1×10⁻⁶ ml/g/sec in six different brain regions (Table1). The PS values for the monoclonal antibody to human Aβ 1-42 (PC2)ranged from 0.6-1.4×10⁻⁶ ml/g/sec in the same brain regions and were notsignificantly different. V_(p) values ranged from 12.8-28.4 μl/g forL227 and from 11.8-28.0 μl/g for PC2 and were not significantlydifferent (Table 1). The PS values for both monoclonal antibodies arelow and less than that observed for albumin. Both IgG and albumin areconsidered to be transported at the BBB by passive diffusion or fluidphase endocytosis. In contrast, insulin has very high PS values in mice(27.7-43.0×10⁻⁶ ml/g/sec) and is transported at the BBB by areceptor-mediated transport. Insulin has a PS product at the BBB that isapproximately 28.3-49.9 fold greater than that of the monoclonalantibody to human Aβ42 (PC2). In contrast, the V_(p) values for insulinand the monoclonal antibody to human Aβ42 (PC2) are similar. TABLE 1 BBBPermeability for the Immune Complex [(hAβ42)-PC2] is Greater than theMonoclonal Antibody Alone (PC2) or a Non-Specific Monoclonal Antibody(L227) (hAβ42)-L227 (hAβ42)-PC2 L227 100:1 PC2 10:1 100:1 1000:1 RI n =7 n = 6 n = 14 n = 6 n = 6 n = 7 100:1 vs PC2 PS: ml/g/sec × 10⁶ Cortex 0.49 ± 0.03  0.95 ± 0.15  0.71 ± 0.10  1.26 ± 0.25  2.87 ± 0.27*** 2.74 ± 0.31*** 4.0 Caudate-Putamen  0.51 ± 0.05  0.63 ± 0.15  0.64 ±0.05  1.04 ± 0.13*  2.33 ± 0.15***  2.04 ± 0.08*** 3.6 Hippocampus  0.59± 0.05  0.90 ± 0.25  0.70 ± 0.06  1.15 ± 0.32  2.43 ± 0.32***  2.82 ±0.25*** 4.0 Thalamus  0.70 ± 0.06  1.05 ± 0.24  0.81 ± 0.06  1.54 ±0.19*  3.21 ± 0.17***  3.09 ± 0.31*** 4.0 Brain Stem  1.10 ± 0.05  1.84± 0.30  1.38 ± 0.15  2.70 ± 0.48*  4.25 ± 0.31***  4.20 ± 0.42*** 3.1Cerebellum  0.82 ± 0.05  1.30 ± 0.19  0.98 ± 0.10  2.36 ± 0.59*  3.58 ±0.24***  3.89 ± 0.54*** 4.0 V_(p): μl/g Cortex 21.36 ± 1.73 26.62 ± 1.5220.07 ± 1.14 24.97 ± 0.36 24.70 ± 3.00 25.60 ± 1.89 1.2 Caudate-Putamen12.77 ± 1.45 16.15 ± 1.61 11.78 ± 0.57 17.73 ± 1.99* 17.54 ± 1.70 17.60± 1.98* 1.5 Hippocampus 20.52 ± 2.09 25.31 ± 3.05 22.51 ± 0.91 27.58 ±1.84 26.06 ± 3.28 25.94 ± 2.50 1.2 Thalamus 18.47 ± 1.15 25.84 ± 2.7017.37 ± 0.98 23.17 ± 1.28* 26.88 ± 3.37** 23.13 ± 2.34 1.6 Brain Stem25.14 ± 1.63 29.10 ± 2.45 23.68 ± 1.72 30.34 ± 1.47* 31.11 ± 2.80 22.79± 1.44 1.3 Cerebellum 28.43 ± 1.99 34.86 ± 2.36 27.99 ± 1.85 37.32 ±1.96 33.84 ± 3.87 30.73 ± 2.36 1.2 {overscore (X)} ± SEM L227: ATCC HB96(Anti-human Ia) IgG₁κ; BALB/c PS: Permeability coefficient × Surfacearea product PC2: MoAb (Anti-human Aβ42) IgG₁κ; BALB/c V_(p): ResidualPlasma Volume RI: Relative increase of immune complex [(hAβ42)-PC2] vs.MoAb (PC2) at mole ratios of 100:1 (hAβ42)-L227 {close oversize brace}Immune complex at mole ratios of 10:1, 100:1, or 1000:1 (hAβ42)-PC2Analysis of variance followed by Bonferroni multiple comparisons; onlysignificant differences shown; *P < 0.05, **P < 0.01, **P < 0.001

[0053] Permeability of immune complexes of human Aβ42 with itsradioiodinated monoclonal antibody at various mole ratios were assessedas described above. At a mole ratio of 10:1 [(human Aβ42)-PC2], asignificant increase in the PS at the BBB in four of six brain regionswas observed compared with the PS values observed for PC2 alone (Table1). When the mole ratio was increased to 100:1, highly significant PSvalues (2.3-4.3×10⁻⁶ ml/g/sec) were obtained in all brain regions(P<0.001). This represents a 3.1 to 4.0-fold increase in the PS values.In contrast, when human Aβ42 was incubated with the non-specificmonoclonal antibody (L227) at the same mole ratio of 100:1, the PSvalues obtained were not significantly different from that in theabsence of the antigen (Table 1). When human Aβ42 was incubated with PC2at a mole ratio of 1000:1, there was a non-significant decrease in thePS values for most of the brain regions indicating that the receptor forhuman Aβ42 at the BBB was beginning to be saturated (Table 1). Incontrast, the V_(p) values showed a slight trend toward being increasedfor the different mole ratios of immune complex compared to themonoclonal antibody, and this reached significance in only a few cases.These studies demonstrate that the BBB permeability for the immunecomplex of (human Aβ42)-PC2 is greater than the monoclonal antibodyalone or the non-specific monoclonal antibody. This suggests that themechanism by which this antibody is crossing the BBB likely involves areceptor for human Aβ at the BBB.

Example 3 Permeability of Polyamine Modified Antibody at the BBB

[0054] In the following series of experiments, PS values ranging from21.5-33.0×10⁻⁶ ml/g/sec in six different brain regions (Table 2) wereobserved for a polyamine modified monoclonal antibody to human Aβ (PC2).These PS values for PUT-PC2 were highly significant (P<0.0001) andranged from 22.8-37.9 fold higher than the antibody (PC2) alone.Polyamine modification of the monoclonal antibody may allow for betterdelivery across the BBB. This approach is not dependant upon circulatingAβ levels and may allow for a more dramatic reduction in amyloid burdenin the Alzheimer brain following passive immunization. TABLE 2 BBBPermeability of Polyamine-Modified Monoclonal Antibody (PUT-PC2) isGreater than the Monoclonal Antibody Alone (PC2) PC2 PUT-PC2 n = 14 n =15 P RI PS: ml/g/sec × 10⁶ Cortex  0.7 ± 0.1 25.1 ± 1.5 **** 35.9Caudate-Putamen  0.6 ± 0.1 21.5 ± 1.4 **** 35.8 Hippocampus  0.6 ± 0.126.5 ± 1.8 **** 37.9 Thalamus  0.8 ± 0.1 27.1 ± 1.6 **** 33.9 Brain Stem 1.4 ± 0.2 31.9 ± 3.3 **** 22.8 Cerebellum  1.0 ± 0.1 33.0 ± 2.3 ****33.0 V_(p): μl/g Cortex 20.1 ± 1.1 17.9 ± 0.7 ns 0.9 Caudate-Putamen11.8 ± 0.6  9.8 ± 0.4 * 0.8 Hippocampus 22.5 ± 1.0 18.3 ± 1.0 ns 0.8Thalamus 17.4 ± 1.0 17.0 ± 0.8 ns 1.0 Brain Stem 23.7 ± 1.7 21.9 ± 1.2ns 0.9 Cerebellum 28.0 ± 1.9 23.7 ± 0.8 ns 0.8

Other Embodiments

[0055] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 1 1 43 PRT Homo sapiens 1 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr GluVal His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly SerAsn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile AlaThr 35 40

What is claimed is:
 1. A composition comprising an amyloid β (Aβ)polypeptide and a non-Aβ polypeptide, wherein said Aβ polypeptide andsaid non-Aβ polypeptide are linked.
 2. The composition of claim 1,wherein said composition further comprises a pharmaceutically acceptablecarrier or excipient.
 3. The composition of claim 1, wherein said non-Aβpolypeptide is an antibody.
 4. The composition of claim 3, wherein saidantibody comprises a Fab fragment.
 5. The composition of claim 3,wherein said antibody comprises a single chain Fv antibody fragment. 6.The composition of claim 3, wherein said antibody comprises a F(ab)₂fragment.
 7. The composition of claim 3, wherein said antibody hasspecific binding affinity for amyloid.
 8. The composition of claim 3,wherein said antibody is labeled with a radioisotope or a contrastagent.
 9. The composition of claim 3, wherein said antibody is labeledwith a contrast agent.
 10. The composition of claim 1, wherein saidnon-Aβ polypeptide is an enzyme or a cytokine.
 11. The composition ofclaim 10, wherein said enzyme is an antioxidant enzyme.
 12. Thecomposition of claim 11, wherein said antioxidant enzyme is catalase orsuperoxide dismutase.
 13. The composition of claim 1, wherein saidnon-Aβ polypeptide is leptin.
 14. The composition of claim 10, whereinsaid cytokine is an interferon or an interleukin.
 15. The composition ofclaim 10, wherein said cytokine is a neurotrophic factor.
 16. Thecomposition of claim 1, wherein said Aβ polypeptide and said non-Aβpolypeptide are covalently linked.
 17. The composition of claim 1,wherein said Aβ polypeptide comprises residues 1-40, 1-42, or 1-43 ofSEQ ID NO:1.
 18. A method of treating a patient diagnosed withAlzheimer's disease, said method comprising administering to saidpatient an amount of a composition effective to treat Alzheimer'sdisease, said composition comprising an Aβ polypeptide and an antibodyhaving specific binding affinity for said Aβ polypeptide.
 19. The methodof claim 18, wherein said antibody comprises a Fab fragment.
 20. Themethod of claim 18, wherein said antibody comprises a single chainFv-antibody fragment.
 21. The method of claim 18, wherein said antibodycomprises a F(ab)₂ fragment.
 22. A method of treating a patientdiagnosed with Alzheimer's disease, said method comprising administeringto said patient an amount of an antibody effective to treat Alzheimer'sdisease, wherein said antibody is polyamine modified and has specificbinding affinity for an Aβ polypeptide.
 23. A method of diagnosingAlzheimer's disease in a patient, said method comprising a)administering a composition to said patient, wherein said compositioncomprises an Aβ polypeptide and an antibody having specific bindingaffinity for amyloid, wherein said antibody is labeled, and b) detectingthe presence or absence of said antibody bound to amyloid in the brainof said patient, wherein said patient is diagnosed with Alzheimer'sdisease based on the presence of labeled amyloid in the brain of saidpatient.
 24. The method of claim 23, wherein said detecting stepcomprises diagnostic imaging.
 25. The method of claim 23, wherein saiddiagnostic imaging comprises positron emission tomography,gamma-scintigraphy, single photon emission computerized tomography,magnetic resonance imaging, functional magnetic resonance imaging, ormagnetoencephalography.
 26. The method of claim 23, wherein saiddiagnostic imaging comprises magnetic resonance imaging.
 27. The methodof claim 23, wherein said amyloid comprises β-amyloid plaques.
 28. Themethod of claim 23, wherein said antibody is labeled with a contrastagent.
 29. The method of claim 28, wherein said contrast agent isselected from the group consisting of gadolinium, dysprosium, and iron.30. The method of claim 28, wherein said contrast agent is gadolinium.